Nitric oxide (NO) was recently discovered to be a cell regulatory molecule with rapid and potent short-range actions in cardiovascular, neuronal and immune systems. NO originates from arginine by an enzymatic reaction involving NO synthase (NOS), for which there are 3 known isoforms. The hypothesis to be tested in this application is that NO (cNOS and iNOS), produced in osteoblasts, is both a direct autocrine regulator of osteoblast proliferation, function and viability, and a paracrine regulator of neighboring bone cell types. NO produced in the bone microenvironment is an important modulator of osteoblastic function. The first Specific Aim is to study the role of pro-inflammatory cytokines in regulating expression of an iNOS, in various osteoblastic cell lines and mutant osteoblasts from NOS knock-out mice using sensitive molecular biological methods. The investigators will search for a cNOS in osteoblasts, purify the NOS enzymes and characterize activators and inhibitors. Through isoform-specific immunostaining of NOS, the pattern of NOS protein expression will be determined in bone cells of transgenic mice deficient in specific NOS isoforms, and in a new mouse model of inflammation-mediated bone loss. The second Specific Aim is to study the mechanisms by which NO regulates osteoblasts, stimulating certain functions at low concentrations, while becoming cytotoxic and triggering osteoblastic death at high concentrations. The investigators will focus on the processes of proliferation, matrix biosynthesis, bioenergetics and apoptosis, searching for effects on putative intracellular target proteins of NO action including ribonucleotide reductase, aconitase, guanyl cyclase, cyclooxygenase and extracellular matrix proteins which can be nitrated by reactive NO intermediates. Finally, cell-cell signaling action of NO will be tested using osteoblast/osteoclast co-cultures. It is hoped that these studies will establish the physiological significance of the NO/NOS system in bone and the extent to which NO modulates normal hormonal regulation of bone physiology. NO originating from osteoblasts may trigger the extensive osteoclastic bone destruction in inflammatory bone diseases, including rheumatoid arthritis, osteomyelitis and periodontal disease, while simultaneously blocking the process of coupling which normal bone uses to maintain bone mass. Strategies to control osteoblastic NO production may prove effective for pharmacologic intervention in a broad range of bone diseases.